A NanoLuc Luciferase Reporter Pseudorabies Virus for Live Imaging and Quantification of Viral Infection.

Yalin Wang,Yuan Sun,Bing Wang,Zhao Jin,Hongxia Wu,Hansong Qi,Hua-Ji Qiu

doi:10.3389/fvets.2020.566446

Abstract

Pseudorabies (PR), also known as Aujeszky's disease, is an acute infectious disease of pigs, resulting in significant economic losses to the pig industry in many countries. Since 2011, PR outbreaks have occurred in many Bartha-K61-vaccinated pig farms in China. The emerging pseudorabies virus (PRV) variants possess higher pathogenicity in pigs and mice than the strains isolated before. Here, a recombinant PRV (rPRVTJ-NLuc) stably expressing the NanoLuc (NLuc) luciferase fusion with the red fluorescent protein (DsRed) was constructed to trace viral replication and spread in mice. Moreover, both DsRed and NLuc luciferases were stably expressed in the infected cells, and there was no significant difference between wild-type and recombinant viruses in both growth kinetics and pathogenicity. Seven-week-old BALB/c mice were infected with 103 50% tissue culture infective dose rPRVTJ-NLuc and subjected to daily imaging. The mice infected with rPRVTJ-NLuc displayed robust bioluminescence that started 4 days postinfection (dpi), bioluminescence signal increased over time, peaked at 5 dpi, remained detectable for at least 6 dpi, and disappeared at 7 dpi, meanwhile, the increased flux accompanied by the spread of the virus from the injection site to the superior respiratory tract. However, the signal was also observed in the spinal cord, trigeminal ganglion, and partial region of the brain from separated tissues, not in living mice. Our results depicted a new approach to rapidly access the replication and pathogenicity of emerging PRVs in mice.

Highlights

Pseudorabies (PR), a devastating disease in the pig industry worldwide, is characterized by neurological signs, respiratory signs, high morbidity, and mortality of piglets, whereas older pigs mostly exhibit respiratory and reproductive diseases [1]
The transfer vector pOK-DsRed-NLuc with two 1.5-kb homologous arms and PRVTJ genomic DNA were co-transfected into Vero cells, and the first generation of recombinant virus was collected at 2–3 days after transfection when the cytopathic effect formed on the Vero cells
The stability of the reporter genes, NLuc, and DsRed, was tested by following amplification and serial passages in PK-15 cells (P1 to P20) (Figure 1D). These results indicated there were glycoprotein B (gB) and glyco-protein I (gI) genes in the genome backbone of the recombinant pseudorabies virus (PRV), which was the same as its parental strain

Summary

Introduction

Pseudorabies (PR), a devastating disease in the pig industry worldwide, is characterized by neurological signs, respiratory signs, high morbidity, and mortality of piglets, whereas older pigs mostly exhibit respiratory and reproductive diseases [1]. Pseudorabies virus (PRV) is the causative agent of the acute infectious PR in swine. The disease is sporadic in many other countries, including China [2]. In late 2011, there was a PR outbreak on many large pig farms where piglets were vaccinated with Bartha-K61vaccine, and quickly the disease occurred in six provinces in China. The mortality rate of infected piglets was from 10 to 50%, which caused great economic loss [3].

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Conclusion